Loline alkaloid gene clusters of the fungal endophyte neotyphodium uncinatum

Abstract

Loline alkaloids (LA), which are 1-aminopyrrolizidines with an oxygen bridge, are produced by Epichloë (anamorph= Neotyphodium ) species, endophytes of grasses. LA are insecticidal, thus helping protect host plants from insect herbivory. Suppression subtractive hybridization PCR was used to isolate transcripts up-regulated during loline alkaloid production in cultures of Neotyphodium uncinatum . Subtracted cDNAs were cloned, and a λ-phage cDNA library from an LA-expressing N. uncinatum culture was screened with subtracted cDNA. In BLAST searches, several cDNAs identified had sequence similarities to aspartate kinases, and another with O-acetylhomoserine-(thiol)lyase. Differential expression of these two genes in LA-producing cultures of N. uncinatum was confirmed, and in a survey of 23 isolates from 21 Neotyphodium and Epichloë species these two genes strictly correlated with LA production. Two nucleic acid molecules encoding two loline alkaloid gene clusters have been identified.

1. An isolated nucleic acid molecule comprising: a nucleotide sequence consisting of or complementary to ORF8 of SEQ ID NO: 15 or a nucleotide sequence encoding a variant which hybridizes under high stringency conditions of 0.1×SSC, 0.1% SDS at 65° C. to ORF8 of SEQ ID NO: 15 and wherein the encoded polypeptide of said variant has γ-type pyridoxal phosphate (PLP) enzyme activity. 
     
     
       2. The isolated nucleic acid of  claim 1 , wherein nucleotide sequence is ORF8 which encodes lolC in SEQ ID NO: 15. 
     
     
       3. An expression vector comprising the nucleic acid compound of  claim 1 . 
     
     
       4. An isolated host cell transformed with an expression vector of  claim 3 . 
     
     
       5. The host cell according to  claim 4  wherein the host cell is a bacterium. 
     
     
       6. The host cell according to  claim 5  wherein the host cell is  E. coli.    
     
     
       7. The host cell according to  claim 4  wherein the host cell is  Neotyphodium.